Protein kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins (Hunter, T., Cell 50 (1987) 823-829). Protein kinases are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain binds and orients the ATP (or GTP) donor molecule. The larger C terminal part binds the protein substrate and carries out the transfer of phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue.
The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contain specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie, G., and Hanks, S., The Protein Kinase Facts Books I, Academic Press, San Diego, Calif., 1995, pp. 7-20).
The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP) cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic ADP ribose, arachidonic acid and diacylglycerol. Cyclic -AMP dependent protein kinases (PKA) and mitogen-activated protein kinases (MAPK) are e.g. members of the STK family. Cyclic -AMP is an intracellular mediator of hormone action in all procaryotic and animal cells that have been studied. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cyclic -AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease.
MAP kinases like p38 also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S. E., and Weinberg, R. A., Nature 365 (1993) 781-783).
Protein kinase B (PKB/Akt) is a component of an intracellular signalling pathway of fundamental importance that functions to exert the effects of growth and survival factors, and which mediates the response to insulin and inflammatory signals (Datta, S. R., et al., Genes Dev. 13 (1999) 2905-2927; Brazil, D. P., and Hemmings, B. A., Trends Biochem. Sci. 11 (2001) 657-664). The recombinant production and purification of PKB is described in WO 2003/016516 using Phenyl TSK hydrophobic interaction chromatography. PKB was adsorbed to the column and eluted after washing PKB using a linear gradient.
Src kinases are implicated in cancer, immune system dysfunction and bone remodeling diseases. For general reviews, see Thomas, S. M., and Brugge, J. S., Annu. Rev. Cell Dev. Biol. 13 (1997) 513-609. Members of the Src family are e.g. Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, and Blk. These are nonreceptor protein kinases that range in molecular mass from 52 to 62 kD. All are characterized by a common structural organization that is comprised of six distinct functional domains: Src homology domain 4 (SH4), a unique domain, SH3 domain, SH2 domain, a catalytic domain (SH1), and a C-terminal 15 regulatory region.
In prokaryotic organisms, the protein synthesis, also referred to as translation, takes place on the ribosomes in the cytoplasm. In expressing recombinant DNA in prokaryotic host organisms, such as, e.g., E. coli the resultant recombinant gene product/protein often precipitates in the cytoplasm in the form of insoluble inclusion bodies. After completion of fermentation and lysis of the cells, the inclusion bodies are isolated and optionally purified and the recombinant protein contained therein is solubilized by adding denaturants such as urea or guanidinium hydrochloride and naturation of said protein is accomplished by reducing the denaturing conditions. Such methods are well-known and have long been used successfully also for the industrial manufacture of recombinant proteins (cf., e.g., Lee, S. Y., Trends Biotechnol. 14 (1996) 98-105; Panda, A. K., et al., J. Biotechnol. 75 (1999) 161-172; Mattes, R., Semin. Thromb. Hemost. 27 (2001) 325-336; Clark, E. D., Curr. Opin. Biotechnol. 12 (2001) 202-207; Misawa, S., and Kumagai, I., Biopolymers 51 (1999) 297-307; and Lilie, H., Current Opinion Biotechnol. 9 (1998) 497-501).
However, expression of mammalian proteins in microbial host cells like E. coli is often a challenging task due to poor solubility, improper folding, lack of stability and other problems. Past attempts to produce such protein kinases by recombinant expression in microbial host cells pursuant to known methods in the art generally result in general only low amounts of active soluble kinases but with large amounts of undesired and inactive dimers and higher aggregates.
Now it was surprisingly found, that using the method of the invention kinases can be recovered after recombinant production in microbial host cells in a correctly folded form in large amounts.